Alkylating agents are among the most effective therapeutic agents currently available to treat different malignancies, and are widely used in the clinic (Katzung, In Basic & Clinical Pharmacology, 7th edition, 1998, Appleton & Lange, Stamford, 881). The high degree of cytotoxicity is attributed to the ability to induce DNA interstrand cross-linking thereby inhibiting replication (Rajski and Williams, Chem Reviews 1998, 98: 2723). Among the alkylating agents, the CNU (chloroethylnitrosourea) series have been widely used clinically to treat brain tumors, colon cancer and lymphomas (DeVita, et al. Cancer Res. 1965, 25: 1876; and Nissen, et al. Cancer 1979, 43: 31), however, their clinical usefulness is limited due to delayed and cumulative bone marrow depression and hepatic toxicity (Panasci, et al. Cancer Res. 1977, 37: 2615; and Gibson and Hickman, Biochem Pharmacol. 1982, 31: 2795).
A series of 1,2-bis(sulfonyl)hydrazine prodrugs (SHPs) with the ability to generate chloroethylating and carbamoylating species, but lacking hydroxyethylating and vinylating species, generated by the CNUs had been developed recently (Sartorelli, et al. see U.S. Pat. No. 6,040,338; U.S. Pat. No. 5,637,619; U.S. Pat. No. 5,256,820; U.S. Pat. No. 5,214,068; U.S. Pat. No. 5,101,072; U.S. Pat. No. 4,849,563; and U.S. Pat. No. 4,684,747). The antitumor activity has been suggested to result from chloroethylating and subsequent cross-linking of DNA (Kohn, In Recent Results in Cancer Research, Eds. Carter, et al., 1981, Springer, Berlin, vol. 76: 141; and Shealy, et al., J Med Chem. 1984, 27: 664). The carbamoylating species (i.e., the isocyanate) can react with thiol and amine functionalities on proteins and inhibit DNA polymerase (Baril, et al. Cancer Res. 1975, 35: 1), the repair of DNA strand breaks (Kann, et al. Cancer Res. 1974, 34: 398) and RNA synthesis and processing (Kann, et al. Cancer Res. 1974, 34: 1982). However, hydroxyethylation of DNA is a carcinogenic and/or mutagenic event (Swenson, et al. J Natl Cancer Inst. 1979, 63: 1469).
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylaminocarbonyl) hydrazine (VNP40101M, Cloretazine™), the current lead compound in the SHP series, has lower toxicity to hosts and better anti-tumor activities against the L1210 murine leukemia, L1210/BCNU, L1210/CTX, L1210/MEL (1,3-bis(2-chloroethyl)-1-nitrosourea, cyclophosphamide and melphalan resistant sublines), P388 leukemia, M109 lung carcinoma, B16 melanoma, C26 colon carcinoma and U251 glioma than chloroethylnitrosourea (CNU) derivatives and other SHP analogs (Shyam, et al. J Med Chem. 1999, 42: 941). In addition, VNP40101M is effective in crossing the blood brain barrier (BBB) and eradicating leukemia cells implanted intracranially (>6.54 log cell kill), rivaling the efficacy of BCNU (Finch, et al. Cancer Biochem Biophys. 2001, 61: 3033).

The anti-tumor activity of VNP40101M is probably due to the release of 90CE and methyl isocyanate. 90CE further fragments to yield methyl 2-chloroethyldiazosulfone (1), see FIG. 1 of U.S. Pat. No. 6,855,695, a relatively specific O6-guanine chloroethylator, producing minimal alkylation of the N7-position of guanine (Penketh, et al. J Med Chem. 1994 , 37: 2912; and Penketh, et al. Biochem Pharmacol. 2000, 59: 283). Methyl isocyanate released from VNP40101M has the ability to inhibit various DNA repair enzymes including O6 -alkylguanine-DNA alkyltransferase leading to stabilization of the O6-alkylguanine monoalkyl species in DNA, which leads to a larger percentage of interstrand cross-links (Baril, et al. Cancer Res. 1975, 35: 1)
Activity in Murine Tumor Models
VNP40101M has shown broad anti-tumor activity against leukemia and solid syngeneic and human xenograft tumors in murine models (Shyam et el., J Med Chem 28:525-7, 1985; Shyam et al., J Med Chem 29:1323-5, 1986; Shyam et al., J Med Chem 30:2157-61, 1987; Shyam et al., J Med Chem 36:3496-502, 1993; Shyam et al., J Med Chem 39:796-801, 1996). The data is summarized briefly below:                1. Against intraperitoneal (IP) implanted L1210 leukemia (106 tumor cells) that is resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide, or melphalan, a single dose of VNP40101M administered IP 24 hours after tumor inoculation (20-60 mg/kg, or 60-180 mg/m2) was curative in 100% of mice (survival ≧60 days) . The VNP40101M doses required to produce long-term survival in sensitive and resistant L1210-bearing mice were only modestly myelosuppressive when administered to non-tumor-bearing mice. Higher single doses (80-100 mg/kg) produced signs of a wasting condition and death in some animals, which occurred >50 days after treatment. When administered IP daily for 6 days, VNP40101M at a dose of 6 mg/kg/d (total dose 36 mg/kg) increased life span of treated mice by 333% compared to control mice, and at doses of 12-18 mg/kg/d (total doses of 72-108 mg/kg) was curative in 100% of animals. Delayed toxic deaths were not observed with the d×6 schedule, however, total doses higher than 108 mg/kg were not tested.        2. Against subcutaneous tumors of the human U251 glioma staged to ˜300 mg in nude mice, VNP40101M administered as 10 mg/kg q2d×11 or 20 mg/kg q4d×6 IP caused complete regressions.        3. Against the murine M109 lung carcinoma implanted subcutaneously and staged to ˜150 mg, VNP40101M administered as 10 mg/kg q2d×10 or 20 mg/kg q4d×5 IP delayed tumor growth, with the latter schedule delaying time to reach 1 gram by 14 days.        4.Administration of VNP40101M weekly by the IP route produced significant delays in tumor growth against the syngeneic B16F10 melanoma and against the human HTB177 lung (H460) and WiDr colon carcinoma cell lines. Doses from 10-60 mg/kg were active, but higher doses produced greater growth inhibition.        5. Substantial anti-tumor activity was observed by IP administration of VNP40101M against intra-cranially implanted L1210 leukemia, demonstrating good penetration through the blood-brain barrier.Toxicology Studies        
The relationship between VNP40101M dose and white blood cell count (WBC) was examined in normal CD2F1 mice. Modest leukopenia (50% of baseline) was observed with a single IP dose of 40 mg/kg (120 mg/m2). VNP40101M doses of 60-80 mg/kg reduced WBC counts to approximately 25 and 15% of baseline, respectively, by day 4 with full recovery by day 21. As noted in section 1.2.A, high single doses of 80-100 mg/kg administered IP to tumor-bearing mice produced signs of a wasting condition and deaths occurring >50 days after treatment.
Toxicology studies were performed in rats. A dose of 3 mg/kg (18 mg/m2), when given intravenously (IV) on a d×5 dosing schedule, produced no clinical signs or symptoms on day 15, but 2/10 rats had lung findings on day 29, including a small amount of thoracic cavity fluid and failure of the lung to collapse. Microscopic findings at the 3 mg/kg (d×5) dose level were primarily limited to the lung and included alveolar edema, congestion, alveolar histiocytosis, and vascular thrombi. The higher dose of 10 mg/kg (60 mg/m2) d×5 produced few significant gross necropsy findings on day 15, but thoracic cavity fluid was found in approximately 50% of animals sacrificed on day 29 and 6/6 animals sacrificed on days 30/31. Histopathologic findings in the lung were similar to those observed at the 3 mg/kg dose level. For doses as high as 10 mg/kg d×5, myelosuppression was not observed. Effects on serum chemistries were limited to decreases in total protein and albumin, which were observed on day 29 in the 10 mg/kg dose group.
In toxicology studies performed in dogs, single doses of 1, 3, 10, and 30 mg/kg were administered intravenously. The 1 and 3 mg/kg doses were well-tolerated and produced minimal clinical signs through at least 21 days of observation. The higher doses of 10 and 30 mg/kg (200 and 600 mg/m2 , respectively) produced marked clinical signs, as well as laboratory abnormalities including increased alkaline phosphatase, decreased albumin, increased bilirubin, increased creatine phosphokinase, and decreased white and red blood cell counts. Toxicity was also assessed for 0.3, 1 and 3 mg/kg doses administered intravenously daily×5. The 3 mg/kg d×5 dose produced marked clinical signs including reduced activity, loose stool, anorexia and slight dehydration, requiring sacrifice of the animals on day 8. There was also marked leukopenia by day 8, and slight elevation of the alkaline phosphatase. A dose of 1 mg/kg (20 mg/m2) d×5 produced minimal clinical signs and symptoms, and only a mild leukopenia on day 8 that recovered to baseline by day 15.
Phase I Studies of VNP40101M
VNP40101M has been studied in two phase I trials conducted in patients with advanced solid tumors or hematologic malignancies. In the first phase I trial, 26 patients with solid tumors were treated by IV infusion over 15-30 minutes at dose levels ranging from 3-305 mg/m2 every 4-6 weeks. The maximum tolerated dose (MTD) was 305 mg/m2. Among the seven patients treated at the MTD, six developed grade 3 thrombocytopenia. The platelet nadir occurred between days 25-33. Five of the patients treated at the MTD developed ≧grade 2 granulocytopenia, but only one patient had a grade 3 event. Hematologic toxicities recovered to ≦ grade 1 between days 32-45. No dose-limiting non-hematologic toxicities were observed.
A second phase I trial is being conducted at the MD Anderson Cancer Center in patients with advanced hematologic malignancies. Twenty-eight patients with relapsed or refractory leukemia (20 acute myeloid leukemia [AML], 3 myelodysplasia, 1 chronic myeloid leukemia in blast crisis, 3 acute lymphocytic leukemia, 1 chronic lymphocytic leukemia) have been accrued to the study at doses ranging from 220-708 mg/m2. Through the dose of 708 mg/m2, no dose-limiting non-hematologic toxicities were observed. At doses ≧400 mg/m2, patients developed a transient infusion-related syndrome consisting of headache, nausea, vomiting, myalgias/cramps, facial flushing, dizziness, tachycardia, and hypotension. The infusion-related reaction was self-limited and resolved within several hours after completing treatment in all patients. Among the seven patients treated at 708 mg/m2, one patient developed prolonged marrow aplasia (>80 days) without evidence of leukemia. Thus, myelosuppression may be a dose-limiting toxicity (DLT) at 708 mg/m2. An additional cohort of patients is currently being evaluated at an interim dose of 600 mg/m2.
Evidence of anti-tumor activity was observed in patients with advanced hematologic malignancies. One previously untreated patient with high-risk myelodysplasia developed a complete response by day 28 after a single course of VNP40101M administered at 300 mg/m2. Although no other patient achieved complete remission, VNP40101M reduced peripheral blood blasts at least transiently in most patients at all dose levels. In addition, a heavily pre-treated patient with AML had substantial clearing of marrow blasts and resolution of gingival leukemic infiltration at a dose of 220 mg/m2, and a patient with AML treated at 532 mg/M2 had reduction in marrow blasts and improvement of neutrophil counts by day 28. The level of activity warrants further exploration of VNP40101M alone and in combination in patients with AML.